What are PAHs?
Polycyclic aromatic hydrocarbons (PAHs) are a class of organic compounds produced by incomplete combustion of complex organic molecules. PAHs are ubiquitous in the environment (air, soil, food, water, etc.) because they are commonly released in car exhaust or by burning fossil fuels and trash. In general, a PAH consists of at least three fused aromatic (benzene) rings, with no heteroatoms or substituents. There are over 100 identified PAHs. Most are highly toxic because they form reactive metabolites and/or derivatives that can be potent mutagens; many are known carcinogens. More information on the toxicity of PAHs can be found on the EPA website or from the Agency for Toxic Substances and Disease Registry (ATSDR) here.
Some Common PAHs
Case Study
EKG was approached by a client to assess their liposomal drug formulation for a number of PAHs down to 5 ng/mL (5 ppb). Liposomes have hydrophilic heads and hydrophobic tails, forming a lipid bilayer. In aqueous solutions, such as this pharmaceutical matrix, they form spherical vesicles that can tightly encapsulate organic compounds, including the client’s drug substance. Of course, PAHs would also be trapped within the liposomes and delivered with the drug substance. Because of these low limits and the intense challenge of this matrix, EKG knew quite a bit of effort developing a method would be required prior to being ready to validate.
Method Development
Before tackling the client’s matrix, we prepared standards of a mixture of 15 PAHs at 100 ppb levels to optimize a chromatographic method. Solutions were analyzed using High Performance Liquid Chromatography (HPLC) with Fluorescence Detection (FLD).
Next, we optimized mobile phase composition and the gradient method (two mobile phases: one organic, one a basic aqueous solution). Working off of some literature values, we then, in sync with our mobile phase gradient, developed a wavelength switching method for the FLD, optimizing both the excitation and emission wavelengths individually for almost every PAH in solution. By preparing lower and lower levels of PAHs in solution, we were seeing limits of detection <1 ppb (1 µg/L).
Example Chromatograph
After we were confident in the analytical capabilities of our HPLC/FLD method, we began spiking PAH mixtures into the drug formulation. We evaluated the composition of our liquid-liquid extraction solvent, finding that recovery was heavily dependent on obtaining a precise ratio of organic-to-aqueous extraction solution. We also had to balance the volume of extraction-solution-to-sample to maintain high in-sample PAH concentrations. In order to extract the PAHs out of the liposomes and into solution, samples were placed on a shaker overnight. We then found a filtration procedure that removed the liposomes from the extraction solution while maximizing recoveries of the extracted PAHs.
Method Validation
Once we had both the sample preparation and the LC method developed, we were ready to validate the method.
- We analyzed a blank, unspiked drug product, and spiked drug product for Specificity to ensure no interfering peaks. The PAH peaks themselves all had a resolution >3.
- We calculated our Limits of Detection (LODs) by averaging the signal-to-noise ratio (S/N) for each PAH in replicate preparations of 1 ppb standard. Some LODs were as low as 0.02 ppb (the highest was 0.5 ppb).
- We calculated our Limits of Quantitation (LOQs) by averaging S/N of a 5 ppb standard. LOQs ranged from 2 ppb down to 0.1 ppb.
- Accuracy was evaluated by looking at the recovery of PAH spikes into formulation at multiple levels. Precision was evaluated by looking at the reproducibility of these spiked replicate preparations. Accuracy and Precision matched client desires.
- While this validation was performed for a limit test method, preliminary development results suggested strong Linearity, yielding calibration curves with a correlation coefficient >0.999 between at least 1-100 ppb.
EKG Labs was able to far exceed our client’s expectations in the development of this method, validate the method without issue, and continually analyze lots of our client’s pharmaceutical product under the validated method.
About EKG Labs
EKG Labs is an analytical service provider for the medical device and pharmaceutical industries. Our analytical services support regulatory filings, product development, and analytical chemistry investigations. EKG regularly conducts impurity investigations in challenging matrices, developing and validating methods to monitor chemicals of potential concern.
Additional specialty analytical services include: extractables, leachables, a variety of USP testing, biocompatibility (ISO 10993), characterization, and other investigational activities.
If you are experiencing a challenge with development and validation of a difficult method or need analytical services to support your product development, please contact us at 810-354-5229.
